Discovery of metabolic regulators of DNA topology and cellular responses to topoisomerase II inhibition

项目总结/摘要 我的研究目标是了解调节拓扑异构酶II（topo II）的细胞机制 功能和响应Topo II抑制。II型拓扑异构酶，如拓扑异构酶II，是必需的酶， 管理DNA超螺旋结构和十链缠结的DNA链，以促进关键过程， 如转录、DNA复制和染色体分离。就人类健康而言，topo II是一种 非常重要的癌症靶点，并且临床上使用各种化疗药物来利用这些靶点。 topo II能够产生DNA损伤并杀死复制细胞。生物化学和结构研究 提供了关于topo II的“链通道”机制和合成方法的有价值的细节， 抑制剂影响这种活性。然而，该领域缺乏对topo II活性如何调节的明确理解， 根据细胞需要。 为了更好地了解topo II如何与调控途径和DNA损伤反应联系起来，我的目标是 使用S。酿酒酵母模型系统，以探索细胞代谢在调节topo II功能中的作用， 进行无偏筛选，寻找参与对topo II抑制反应的基因。在分析 真核生物拓扑II的进化保守性，我发现了一个高度保守的口袋拓扑II，相互作用 ICRF-187是一种临床批准的合成抑制剂，用于化疗方案2。我后来 发现这个位点也与白藜芦醇有关，白藜芦醇是一种在红葡萄酒中发现的天然产物。这些观察结果 这表明该位点可能是一个孤儿变构位点，并启发了小分子 代谢物可以变构调节Topo II功能。初步数据表明，酵母的成分 代谢物提取物能够调节Topo II活性。目的1概述了天然产物的纯化 策略和基于质谱的代谢物配体筛选，我将采用识别和表征 内源性小分子可能具有拓扑异构酶II调节功能。除了先前的探测之外， 细胞代谢和DNA拓扑结构之间的未探索的联系，我的目标是揭示参与的途径， 对异常拓扑异构酶II活性的细胞反应。因此，在目标2中，我提出了一种无偏倚的药物遗传学筛选方法 与酵母缺失和亚型文库，以确定在临床上存在的情况下影响生长的等位基因， 相关的拓扑异构酶II抑制剂。总之，这些目标有可能发现拓扑结构之间的新的相互作用， 二是调控路径。这样的发现，反过来，可以揭示新的方法来变构调节拓扑异构酶II 联合抗拓扑异构酶抑制剂的组合疗法的功能和新靶点， 有效性和/或降低目前可用治疗的毒性。

Project Summary/Abstract The goal of my research is to understand the cellular mechanisms that regulate topoisomerase II (topo II) function and respond to topo II inhibition. Type II topoisomerases, such as topo II, are essential enzymes that manage DNA superhelical structure and decatenate entangled DNA strands to facilitate critical processes such as transcription, DNA replication, and chromosome segregation1. With respect to human health, topo II is a tremendously important cancer target, and a variety of chemotherapeutics are clinically used to exploit the ability to topo II to generate DNA damage and kill replicating cells. Biochemical and structural studies have provided valuable details about the “strand passage” mechanism of topo II and the means by which synthetic inhibitors affect this activity. However, the field lacks a clear understanding of how topo II activity is regulated in accordance with cell needs. To better understand how topo II is connected to regulatory pathways and DNA damage responses, I aim to use the S. cerevisiae model system to explore the role of cellular metabolism in regulating topo II function and perform an unbiased screen for genes involved in responding to topo II inhibition. While analyzing the evolutionary conservation of eukaryotic topo IIs, I discovered a highly conserved pocket on topo II that interacts with ICRF-187, a clinically approved, synthetic inhibitor that is used in chemotherapeutic regimens2. I later discovered that this site also engages resveratrol, a natural product found in red wine3. These observations suggest that this site may be an orphan allosteric site and inspired the hypothesis that small-molecule metabolites may allosterically regulate topo II function. Preliminary data indicate that components of yeast metabolite extracts are able to modulate topo II activity. Aim 1 outlines the natural product purification strategies and mass spectrometry-based metabolite ligand screening I will employ to identify and characterize endogenous small molecules that may have topo II-regulatory function. In addition to probing previously unexplored connections between cellular metabolism and DNA topology, I aim to uncover pathways involved in cellular responses to abnormal topo II activity. Thus, in Aim 2 I propose an unbiased pharmacogenetic screen with yeast deletion and hypomorph libraries to identify alleles that affect growth in the presence of clinically- relevant topo II inhibitors. Together, these aims have the potential to discover novel interactions between topo II and regulatory pathways. Such discoveries, in turn, could unveil new ways to allosterically modulate topo II function and new targets for combinatorial therapy in conjunction with anti-topoisomerase inhibitors to improve the efficacy and/or decrease toxicity of currently available treatments.